The present inventors (Maeda et al.) have found that styrene-maleic acid (or maleic anhydride) copolymers (hereinafter, referred to as “SMA”) may be an excellent carrier for pharmaceuticals due to the remarkable amphiphilicity. Accordingly, the present inventors developed SMANCS, a useful macromolecular-conjugated carcinostatic agent (that was approved by the Japanese Ministry of Health, Labour and Welfare in 1993; see U.S. Pat. No. 4,182,752; U.S. Pat. No. 4,732,933; CA Patent No. 1241640; EP Patent No. 0136791; H. Maeda et al., Int. J. Peptide & Protein Res 14, 81-87, 1979; H. Maeda, Adv. Drug Deliv. Rev. 46, 169-185, 2001; and K. Greish, H. Maeda, Drug Delivery System, vol. 18, No. 3, p 254, May 2003 (Abstract of presentation at the Japan Society of Drug Delivery System Annual Meeting)).
SMANCS comprises two SMA chains cross-linked to a small protein, neocarzinostatin (NCS; molecular weight (MW): 12 KDa) by reacting two free amino groups in neocarzinostatin (Ala 1 and Lys 20) with a maleic anhydride residue in the SMA chain (comprising 20 to 45% of maleic anhydride residues, wherein the SMA retains a partial half butyl ester). This reaction is for cross-linking NCS, which is only soluble in water, to a maleic anhydride residue in the SMA chain by forming amide bonds in an alkaline aqueous reaction system. This reaction is expected to comprise hydrolysis of maleic anhydride residues in the SMA chain at alkaline pH in the presence of water molecules at a concentration of 55 M and formation of amide bonds between the maleic anhydride residue and amino groups of NCS (2 molecules) on the coexisting NCS molecule at micromolar range of concentrations. In this reaction, most of the maleic anhydride are exhausted and degraded due to the hydrolysis with largely excessive water, thus a largely excessive amount of SMA is required. It results in the difficulties in the separation and purification of the reaction products [Problem 1].
Maleic anhydride in SMA is hardly soluble, in water, but gradually become water-soluble as the ring of the maleic anhydrides is opened by hydrolysis. However, the reaction of SMA with aromatic (aryl) amines, diethylaminated derivatives of protoporphyrin described below, or amino groups on certain peptides proceeds slowly and thus the yield efficiency of the product (complex) is relatively low [Problem 2].
Small-molecule compounds cross-linked to SMA chains as described above form macromolecular compounds (SMA complexes) comprising copolymers (SMA) of hydrophobic units and hydrophilic units as components and thus easily form micelles by themselves. In brief, it has been found that the SMA conjugates are advantageous in that they form self-association type micelles. As an example of the micelles, the present inventors have successfully synthesized micelles composed of complexes of protoporphyrin (protoporphyrin IX, hereinafter referred to as “PP”) and SMA (K. Greish, H. Maeda, Drug Delivery System, vol. 18, No. 3, p254, May 2003; M. Regehly et al., Bioconj. Chem. (2007) vol. 18, 1031-1038; K. Greish, T. Sawa, et al., J. Cont. Release (2004) 97, 219-230; K. Greish, A. Nagamitsu et al., Bioconj. Chem. 16, 230-236, 2005; A. Iyer et al., Biomaterials (2007) vol. 10, 1871-1881; and A. Iyer, K. Greish, T. Seki, S. Okazaki, J. Fang, K. Takeshita, H. Maeda, J. Drug Target 2007, 15, 496-506).
It has been found that PP forms disc-shaped conjugates (aggregates) and the conjugates often form conjugate micelles comprising SMA as a component and conjugating small molecular drugs via a non-covalent bond when the above reaction for binding PP to SMA is performed in an aqueous solution (JP Patent No. 4522452). The SMA-PP conjugate micelles apparently distribute uniformly and normally although they conjugate PP as a noncovalent association. The PP association itself behaves as a micelle of SMA polymers in the aqueous solution. However, it has been found that micelles comprising SMA conjugated to PP via a covalent bond (covalent conjugate) are also formed and coexist (be mixed) with the above micelles comprising the non-covalent conjugated PP in the aqueous solution, and it is thus difficult to separate and purify each micelles [Problem 3].
Therefore, the micelles prepared as described above comprise a mixture of micelles of non-covalent SMA-PP conjugates and micelles of covalent SMA-PP conjugates. When the mixture is administered intravenously, PP in the non-covalent conjugates (micelles) is dissociated from the SMA micelles and released solely. The released PP weakly binds to serum proteins or becomes free PP and thus distributes in liver and/or spleen with high possibilities. It has been found that the decreased amount of PP is moved and accumulated into the targeted lesions, such as tumor sites due to the EPR (Enhanced Permeability and Retention) effect, which is inherent to macromolecular micelles and nanomedicines and thus the risk of hepatic dysfunction is increased (see Cancer Res. 1986, H. Maeda, Adv. Drug Deliv. Rev. 65, 71-79, 2013; H. Maeda, J. Cont. Release 164, 138-144, 2012; and H. Maeda et al., J. Cont. Release 74, 47-61, 2001) [Problem 4].
Further, the conventional SMA micelles are polyanionic and thus the surfaces of the superfine particles have relatively negative charges of −48 to −50 mV. It has been revealed that most of SMA derivatives and micelle particles comprising them accumulate into liver and/or spleen due to the strong negativity (strong negative charge).
Methods for inhibiting the distribution (accumulation) of SMA-derivatives into liver and/or spleen have not been developed so far.